Article Figures & Data

Figures

Cytological parameters of N-resupplied cells. A and B, Size distribution (A) and cell count (B) of the PL and cht7 cells at different times (h) following N resupply. C, Relative DNA content of the PL and cht7 following N resupply. 1C and 2C, one copy and two copies of chromatin content. Five independent biological repeats were examined for (A–C), all showing a similar pattern with one representative result depicted. Results shown here in A and B are from the same experiment. D, Per cell FA content for total lipid and TAG following N resupply of the PL. Averages (n = 3) and sd are indicated. E to H, Transmission electron micrographs showing an overview of wild-type CC-125 cells at the indicated time points (h) during N resupply (NR). The darkly stained vacuole bodies are marked by yellow arrows. I to L, Closer details of subcellular organization of NR12 CC-125 cells. Physical contact of lipid droplets with the chloroplast outer envelope (I); small lipid droplet fused with the vacuole (J); space between thylakoid membranes (K); internal degradation of the unknown vacuole body (L). The scale bar is indicated in each panel. ES, Eyespot; LD, lipid droplet; OE, chloroplast outer envelope; P, pyranoid; SG, starch granule; V, vacuole.

Summary scheme of transcriptomic analyses across different N regimes. A, Graphic illustration of the early-, mid-, and late-reverse gene groups in the PL. Numbers represent the transcripts whose abundance changed according to the N status using a 2-fold cutoff (log2 fold change equals to 1) and a P value < 0.05. +N, N-replete; -N, N-deprived; NR6 and NR12, 6 and 12 h of N resupply, respectively. Numbers associated with the dashed line were inferred. B, Heat map of the overrepresented MapMan categories in the early-, mid-, late-reverse and NR-specific gene groups. The first column has the bin code of the categories and the second column lists the explanation of each category. The color in the heat map represents the -log10 of the q-value obtained from Fisher’s exact test ranging from 1.3 to 10 (0.05–1E-10). The number of genes in each gene group is given in parentheses near the gene group name in the x axis of the heat map. C, Graphic illustration of the NR-specific gene groups in the PL. Numbers represent the transcripts whose abundance did not vary in the –N over +N RNA-seq comparison (−1 < log2 fold change < 1), but differed over 2-fold (P value < 0.05) in both NR over +N and NR over –N comparisons.

Comparative transcriptomics of the PL and the cht7 mutant. A and B, Global gene expression analysis of the PL and cht7 following N resupply. Blue circles: total number of genes changed in expression in a comparison of PL after 48 h of N deprivation followed by 6 h (A) or 12 h (B) of N resupply over PL N-deprived for 48 h. Yellow circles: total number of genes changed in expression in a comparison of PL NR6 over cht7 NR6 (A) or PL NR12 over cht7 NR12 (B). NR6 and NR12, 6 and 12 h of N resupply, respectively. C, Heat map of the overrepresented MapMan categories in the overlapping gene groups as depicted in A and B. The first column has the bin code of the categories and the second column lists the explanation of each category. The color in the heat map represents the -log10 of the q-value obtained from Fishers exact test ranging from 1.3 to 10 (0.05–1E-10). The number of genes in each gene group is given in parentheses near the gene group name in the legend of the heat map.

Gene expression and metabolite level for two selected pathways. A, Overview of the expression of genes involved in the tetrapyrrole pathway (top) and in peroxisomal redox homeostasis (bottom). RNA-seq comparisons of the PL at different N status and the comparisons between PL and cht7 at each N status are shown in the heat map. +N, N-replete; -N, N-deprived; NR6 and NR12, 6 and 12 h of N resupply, respectively. For the genes whose expression pattern following N resupply has been classified, the respective categories are indicated on the right of the heat map. Early, Early-reverse; Mid, mid-reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific. B, Chlorophyll content. C1-C4, 4 independent complemented lines. C, TBARS content. For all quantitative data, averages (n = 3) of biological replicates and sd are indicated.

Lipid analysis and the expression profile of MGDG synthesis genes. A, FA content of total lipid and TAG in the PL following NR at times indicated (h). B, Relative FA compositions of PL and cht7 following N resupply. FAs are designated by the total carbon number followed by the number of double bonds. The position of specific double bonds is indicated either from the carboxyl end “Δ” or from the methyl end “ω.” C, Polar lipid contents in the presence (+N, N-replete) or absence (−N, N-deprived) of N, or following N resupply at times indicated. The y axis is depicted as the ratio of individual polar lipid FAs over total FAs. Averages (n = 4) of biological replicates and sd are indicated. D, Overview of the expression of genes responsible for MGDG synthesis. RNA-seq comparisons of PL at different N status and the comparisons between PL and cht7 at each N status are shown in the heat map. Arrows indicate the sequence of reactants. FAs at the sn-1/sn-2 position of diacylglycerol (DAG) or MGDG are shown.

Gene expression of putative lipase and β-oxidation genes. A, An example depicting how lipases might positively or negatively influence TAG content. MAG, monoacylglycerol; PGD1 and LIP1, lipases. B, Transcript profiles of genes encoding lipases possibly involved in the degradation of TAG (top), in the production of TAG (middle), or in β-oxidation (bottom). RNA-seq comparisons of the PL at different N status and the comparisons between PL and cht7 at each N status are shown in the heat map. +N, N-replete; −N, N-deprived; NR6 and NR12, 6 and 12 h of N resupply. For the genes whose expression pattern following N resupply has been classified, the respective categories are indicated on the right of the heat map. Early, Early-reverse; Mid, mid-reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific.

The effect of the cAMP-PKA pathway on the recovery from N deprivation. A, ELISA assay to quantify the cellular content of cAMP in the PL. +N, N-replete; −N, N-deprived; NR, N resupply at times indicated (h). Asterisks indicate a statistically significant difference (unpaired t test, P < 0.05). B, TAG degradation of PL and cht7 in the presence of PKI. The TAG content (depicted as the ratio of TAG FA over total FA) of cells just before N resupply (designated here as NR0) is shown on the far left. TAG contents of cells treated with different concentrations of PKI (as indicated in the x axis) were quantified at 24 h of N resupply (NR24). C, The regrowth of PL and cht7 in the presence of PKI. The fold change of regrowth is calculated by dividing the cell count measured at 24 h of N resupply by that at 0 h of N resupply (NR24/NR0). PKI treatments in B and C were done simultaneously with N resupply. D, Transcript profiles of genes encoding putative adenylyl cyclase and phosphodiesterase. RNA-seq comparisons of PL at different N status and the comparisons between PL and cht7 at each N status are shown in the heat map. For the genes whose expression pattern following N resupply has been classified, the respective categories are indicated on the right of the heat map. Early, Early-reverse; Mid, mid-reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific. For all quantitative data, averages (n = 3) and sd are indicated.